Kinase related protein (KRP), also called telokin, is an independently expressed "gene within a gene". It represents the carboxyl-terminal 20,000 Da of myosin light chain kinase (MLCK). Its expression is controlled by its promoter which lies within an intron of the MLCK gene. KRP does not contain the enzymatic nor the calmodulin-binding site of MLCK and its function has remained unknown. We have found that it binds to smooth muscle and nonmuscle myosin and may play a role in stabilizing myosin filaments. Unphosphorylated vertebrate smooth muscle and nonmuscle myosin filaments at physiological ionic strengths are depolymerized by the addition of ATP in vitro. The solubilized myosin adopts a "folded" monomeric conformation with a sedimentation coefficient of 10S. KRP prevents this depolymerization by binding to myosin, probably near the "neck" region, i.e., the junction of the head and the subfragment-two (S-2) region. The stoichiometry of binding appears to be 1 mol of KRP per mol of myosin. KRP binds to dephosphorylated smooth and nonmuscle myosin, but not to phosphorylated myosin or skeletal muscle myosin. KRP does not appear to activate the enzymatic activity of the dephosphorylated myosin as measured by its effects on the actin-activated MgATPase nor cause the dephosphorylated myosin to move actin filaments in an in vitro motility assay. KRP does compete with MLCK for the binding of myosin to actin, but does not fully inhibit the phosphorylation of myosin by MLCK. There is nearly 1 KRP per myosin molecule in smooth muscle tissue. We propose that KRP functions to stabilize myosin filaments possibly by binding to the same region in the neck region of myosin to which the tail of muscle myosin binds in forming the 10S conformation. This would account for the observation that relaxed smooth muscle tissue contains myosin thick filaments even though there is a very low level of phosphorylation of the light chains.